The essence of this research proposal is the development of human cell lines and methods suitable for the routine production of human hybridomas, comparable to those established for successfully utilized murine systems. The selection of a variant of the human lymphoblastoid cell line WI-L2-729-HF2 which does not produce immunoglobulin (Ig) and retains a high fusion efficiency is our first goal. This step will improve upon the already favorable properties of the WI-L2-729-HF2 system, namely, the rapid production of stable human-human hybridomas at high frequency (greater than 10 to the minus 5) when fused with motigen-stimulated human B cells which secrete high levels (5-30Mug/ml) of new human Ig isotypes (IgA or Lambda) not produced by the parent tumor line, in both conventional and serum-free medium, as demonstrated in this laboratory. At present, WI L2-729-HF2 produces trace levels of endogenous Ig: 50-100ng/m1 of an IgG,K; surface IgM,K; but no detectable IgA or Lambda. An Ig non-producing line will be selected by cloning non-secreting variants from populations enriched for surface Ig-negative cells by panning or FACS methods. Our next goal is to define optimal conditions for generating specific hybridomas from human peripheral blood lymphocytes (PBL), investigating, in particular, in vivo and in vitro immunization procedures and the role of growth and maturation factors in inducing antigen-specific B cells to a stage optimal for fusion with WI-L2-729-HF2. Primary in vitro immunization protocols developed for murine spleen cells which result in high yield of specific hybridomas against weak antigens will be adapted to human PBL, including gliadin, myelin basic protein, and glutaraldehyde-fixed cells as antigens. This step will likely be critical to producing human hybridomas specific for weak tumor antigens. These studies should ultimately allow full exploitation of human hybridomas to provide tools and reagents for the diagnosis and treatment of human autoimmune disease and cancer. The growth of human hybridomas in serum-free medium and immune-deficient mice should result in production of human antibodies in high yield and purity for future clinical utilization.